Lymphatic vessels play a major role in tissue pressure homeostasis, immune responses, and the uptake of dietary fat and fat-soluble vitamins, as well as in inflammation and cancer progression (Cueni and Detmar, 2006). Recent studies indicate that both lymphatic and blood vessels are involved in chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis (Alitalo et al., 2005; Carmeliet, 2003; Cueni and Detmar, 2006). But the formation and activation of both types of endothelium have also important roles in the progression and metastasis of the majority of human cancers (Alitalo et al., 2005; Carmeliet, 2003). Tumors need to induce the growth of new blood vessels (angiogenesis) in order to secure the sufficient supply of oxygen and nutrients. The growth of new lymphatic vessels (lymphangiogenesis) has been shown to promote cancer metastasis to sentinel lymph nodes and beyond (Hirakawa et al., 2007; Hirakawa et al., 2005; Mandriota et al., 2001; Skobe et al., 2001; Stacker et al., 2001), a phenomenon which is also found in human neoplasm (Dadras et al., 2005; Tobler and Detmar, 2006). Indeed, studies have revealed that tumor-induced lymphangiogenesis around the primary neoplasm is the most significant prognostic indicator to predict the occurrence of regional lymph node metastasis in human malignant melanomas of the skin (Dadras et al., 2005). More recently, it has been found that tumors can induce lymphangiogenesis in their draining lymph nodes, even before they metastasize and that induction of lymph node lymphangiogenesis promotes the further metastatic cancer spread to distant sites (Hirakawa et al., 2007; Hirakawa et al., 2005). Thus, tumor-induced lymphatic growth and activation represents a promising target for treating or preventing advanced cancer. As a result, there has been a surge of interest in identifying key players that can be used to specifically target these processes therapeutically.
A strong correlation between the expression levels of the lymphangiogenic factor vascular endothelial growth factor-C (VEGFC), tumor lymphangiogenesis and lymph node metastasis has been found in human and in experimental tumors (Pepper et al., 2003). VEGFC promotes lymphangiogenesis by activating VEGF receptor-2 (VEGFR2) and VEGFR3 on lymphatic endothelial cells (Makinen et al., 2001). VEGF-C-deficient mice fail to develop a functional lymphatic system (Karkkainen et al., 2004), and transgenic expression of a soluble VEGFR-3 results in pronounced lymphedema (Makinen et al., 2001). However, blockade of the VEGF-C/VEGFR-3 axis only partially inhibits lymphatic metastasis, indicating that additional pathways are involved in mediating the formation and growth of lymphatic vessels. There have been previous attempts to identify lymphatic specific receptors and pathways by transcriptional and proteomic profiling of cultured lymphatic endothelial cells (LEC) (Hirakawa et al., 2003; Petrova et al., 2002; Roesli et al., 2008). However, large-scale functional in vivo screens to identify molecular pathways or drug-like small molecule modulators of lymphatic vessel formation have been missing to date.
In the last years, cost-efficient maintenance together with abundant experimental techniques and molecular tools, have made zebrafish the only vertebrate model used for large-scale in vivo drug screens (Zon and Peterson, 2005). Amphibians offer many of the same experimental advantages that have favored zebrafish in the past, such as rapid extra-uterine development, the transparency of developing tadpoles, and the permeability of the skin for small molecules, but they have to date not been employed for large-scale chemical library screens to gain insight into vascular development. Amphibians have a common evolutionary history with mammals that is an estimated 100 million years longer than between zebrafish and mammals (Brändli, 2004). Being both tetrapods, amphibians and mammals share extensive synteny at the level of the genomes and have many similarities in organ development, anatomy, and physiology (Christensen et al., 2008; Raciti et al., 2008). These traits favor the use of amphibians for large-scale in vivo drug screens. In the past, embryos and tadpoles of the African clawed frog (Xenopus laevis) have served as a powerful animal model to study blood vascular development and angiogenesis (Cleaver and Krieg. 1998; Helbling et al., 2000; Kahn et al., 2007; Levine et al., 2003). More recently, Xenopus embryos were shown to develop also a complex, well-defined lymphatic vascular system (Ny et al., 2005). Similar to the development of the mammalian lymphatic vascular system, LECs transdifferentiate from venous blood vascular endothelial cells (BVEC) and lymphangioblasts contribute in Xenopus to newly forming lymph vessels that mature to drain fluids from the peripheral tissues back to the blood circulation. Antisense-morpholino knockdown studies of the lymphangiogenic factor VEGFC in Xenopus embryos causes lymphatic vessel defects similar to the phenotype observed in VEGFC-deficient mice, including impaired LEC sprouting and migration, and the formation of lymphedema (Karkkainen et al., 2004; Ny et al., 2005).
Various publications have described the use of Xenopus embryos in the study of angiogenesis and lymphangiogenesis. For example, U.S. Patent Application Publication No. 2006/0159676 A1 describes methods of inhibiting and promoting various physiological processes, including angiogenesis and lymphangiogenesis by interference with the apelin/APJ signaling pathway, as well as methods of identifying therapeutic agents affecting the apelin/APJ signaling pathway. The reference describes the effects of various treatments on apelin expression in frog embryos, as measured by in situ hybridization, but does not describe an anatomical pattern of edema formation.
U.S. Patent Application Publication No. 2007/0107072 describes transgenic amphibian models for lymphatic vessel development, including assays that allow screening for compounds able to modulate lymphangiogenesis. The reference describes the use of transgenic frog embryos to study the development of the lymphatic vascular network. The reference does not, however, describe an anatomical pattern of edema formation.
There is, therefore, a need for additional methods for in vivo screening in amphibian model systems and for compounds identified using such screens.